WO2004070030A1 - Overexpression vector for animal cell - Google Patents

Abstract

It is intended to provide an overexpression vector which makes it possible to easily acquire a high-level producing strain with the use of mammalian cells, in particular, Chinese hamster ovary cells (CHO cells) as a host. Namely, an expression vector capable of inducing high productivity of a genetically modified protein in animal host cells which comprises a promoter strongly inducing expression, a multicloning site for gene integration and a polyadenylation signal sequence, in this order starting from the upstream, and a drug-resistance gene having no promoter functioning in animal cells in the downstream thereof.

Description

 Specification

 High expression vector for animal cells

 The present invention relates to a DNA cell vector for animal cells that achieves high-level production of a recombinant protein using animal cells as a host. The vector of the present invention is particularly useful for expressing a protein that cannot produce an active protein when using bacteria, eukaryotic microorganisms, and insect cells as hosts. Background art

 Many vectors for recombinant protein production have been developed, and high yields can be expected in expression systems using bacteria, eukaryotic microorganisms, and insect cells as hosts. However, in expression systems using these as hosts, there may be problems with protein modification or loss of characteristics unique to mammalian proteins. Therefore, for example, in the case of a protein whose biological activity depends on a sugar chain, it is necessary to produce the protein using mammalian cells as a host. Despite these demands, the productivity of recombinant protein expression systems using mammalian cells as hosts is generally low, and there are often problems with the stability of the transgene. Examples of the production of a recombinant protein using mammalian cells as a host include erythropoietin (Table 2), IFN- / 3 (JP-A-7-265580) ), Etc. Also, there have been many reports on the production of recombinant monoclonal antibodies (Japanese Patent Application Laid-Open Nos. Hei 7-67648, Hei 6-30708, Hei 6-21718). 8 6). Disclosure of the invention

Improving the productivity of recombinant proteins using mammalian cells as a host is not only very important in the production of therapeutic drugs and diagnostics, but also greatly contributes to the development and research of them. Therefore, a vector that enables easy acquisition of high-level producing strains using mammalian cells, especially Chinese hamster ovary cells (CH0 cells) as a host, has been developed. The development of tar is urgently needed.

 In order to be able to easily obtain a high-level producing strain, it is only necessary that more high-level producing strains of the target substance be present in fewer transformed cells. This facilitates selection of high level productive clones.

 Gene expression levels have been found to vary significantly with chromosomal location (Annu, Rev. Cell Biol., 6, 679, 1990). In order to obtain a high-level producing strain, it is considered important to target to the high level and high level of gene expression on the chromosome with the quality and performance of the vector DΝII used for transformation.

 As described above, the problem to be solved by the present invention is to provide an expression vector that can easily obtain a high-level productivity strain using mammalian cells, particularly Chinese hamster ovary cells (CH0 cells) as a host. To provide.

 The present inventors have developed a vector having a mechanism in which a gene cassette of interest is integrated into a host cell chromosome at a position where the level of gene expression is high, and as a result, a neomycin resistant strain survives. It is important to minimize the amount of neomycin phosphotransferase (hereinafter referred to as NPT) gene expression in order to enable targeting to high chromosome gene expression levels. In addition, the vector has a dihydrotallowate reductase (hereinafter, referred to as) gene, and by contriving the expression level, efficient gene amplification can be achieved by stimulation with methotrexate. In the present invention, a vector that enables both of the above has been developed. As a result, a vector capable of producing a high-level and stable protein can be produced, and the present invention has been completed.

 That is, according to the present invention, the following inventions are provided.

 (1) Drug resistance that contains, in order from the upstream, a strong expression-inducible promoter, a multicloning site for gene integration, and a polyadduction signal sequence, and that has no downstream promoter operable in animal cells An expression vector containing a gene, which induces high productivity of a recombinant protein in animal host cells.

(2) Strong expression inducible promoter is human cytomegalovirus The expression vector according to (1), which is a Majorlmmeidately-Early antigen promoter, CMV5 promoter (synthetic chimera one promoter), actin promoter or SV40 early promoter.

 (3) the drug resistance gene is a neomycin resistance gene, a codon non-optimized neomycin resistance gene, a hygromycin resistance gene, a zeocin resistance gene, or a plasticidin resistance gene; (1) or (2) 2. The expression vector according to 1.

 (4) The expression vector according to any one of (1) to (3), wherein the drug resistance gene is a neomycin phosphotransferase gene.

 (5) The expression vector according to any one of (1) to (4), wherein the drug-resistant gene is a neomycin phosphotransferase gene derived from Escherichia coli transposon Tn5.

 (6) a minute amount of messenger RNA or protein of the drug resistance gene is generated by the read-through effect due to the presence of the drug resistance gene downstream of the strong expression inducible promoter blocked by the polyadduction signal sequence,

The expression vector according to any one of (1) to (5).

 (7) In addition to the drug resistance 'gene, there is a dihydrofolate reductase gene without a promoter operable in animal cells or a codon non-optimized dihydrofolate reductase gene downstream of the live gene. The expression vector according to any one of (1) to (6).

(8) Downstream of the drug resistance gene, in order from the upstream, contains a strong expression inducible promoter, a multiple cloning site for gene integration, and a polyadenylation sequence, and a promoter operable in animal cells downstream. The expression vector according to any one of (1) to (7), having a dihydrofolate reductase gene or a codon non-optimized (dioptimized) dihydrofolate reductase gene that does not have a gene.

(9) It has a dihydrofolate reductase gene or a codon non-optimized dihydrofolate reductase gene linked downstream of a promoter with low expression inducibility. The expression vector according to any one of (1) to (6).

 (10) A promoter derived from a protein gene whose expression is low in expression is normally expressed only in a very small amount in mammalian cells, a virus antigen gene which is difficult to infect mammals, or an enhancer sequence derived from the above promoter. The expression vector according to any one of (7) to (9), wherein the expression vector is a promoter from which is deleted.

(11) A recombinant expression vector obtained by incorporating a target gene into a multicloning site for gene integration of the expression vector according to any one of (1) to (6).

 (12) A recombinant expression vector obtained by incorporating a target gene into a multicloning site for gene integration of the expression vector according to any one of (7) to (10).

 (13) A transformant having the expression vector according to any one of (1) to (10) or the recombinant expression vector according to (11) or (12).

 (14) A method for producing a transformant that highly expresses a target gene, comprising introducing the recombinant expression vector according to (11) or (12) into a host animal cell.

 (15) A transformant is obtained by introducing the recombinant expression vector according to (11) or (12) into a host animal cell, and the obtained transformant is adapted to a serum-free medium. A method for obtaining a transformant capable of stably producing a protein in a bloodless medium, comprising the step of:

 (16) A method for amplifying a target gene, comprising introducing the recombinant expression vector according to (12) into a CHO cell deficient in dihydrofolate reductase, and then culturing the cell in the presence of methotrexate.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a flow chart for the preparation of the neomycin resistant vector pSV / GKT-Neo. FIG. 2 shows a flow chart for constructing a neomycin resistance _/ methotrexate resistance vector pND.

 FIG. 3 shows a flow chart for preparing a cassette plasmid pCB for desired gene expression.

 FIG. 4 shows a flow chart of the construction of a foreign gene expression vector pNOS-GKT2 having an NPT gene cistron without a transcription promoter active in mammalian cells.

 FIG. 5 shows a flow chart for preparing a foreign gene expression vector pNOS-GKT2B in which bGH-polyA for transcription termination is arranged upstream of the NPT gene cistron without a promoter.

 FIG. 6 shows a flow chart of the production of a foreign gene expression vector pNOS-GKT2B in which a perforation globin gene intron for improving the translation efficiency is arranged.

 FIG. 7 shows a map of the expression vector pNOS-GKT2B of the present invention.

 FIG. 8 shows a flowchart of production of the vector for expressing hG-CSF, pNOS-GKT2B-R / hG-CSF.

 FIG. 9 shows an example of detection of hG-CSF expressed by a clone obtained by transfection of the expression vector pNOS-GKT2B of the present invention by dot blotting.

 FIG. 10 shows the distribution of the expression level of hG-CSF in each clone transfected with the expression vector pNOS-GKT2B of the present invention.

FIG. 11 shows a map of the expression vector pNOS of the present invention used in Example 4. FIG. 12 shows an example of detection of hG-CSF expressed by a clone transfected with the expression vector pNOS / human G-CSF of the present invention by dot blotting. FIG. 13 shows an example of detection of hG-CSF expressed by a clone obtained by transfection of the expression vector-pNOS / human G-CSF of the present invention by dot blotting. Figure 1 4 shows a hG- expression distribution of the amount of CSF in each clone the expression base Kuta one _P N0S / h丽n G-CSF was transflector Ekushi Sauvignon present invention.

FIG. 15 shows a flowchart of the production of the CMV5 promoter. FIG. 16 shows a flowchart of the production of the CMV5 promoter.

 FIG. 17 shows a flowchart of the production of pCMV5-luciferase.

 FIG. 18 shows a comparison of the activity of the CMV promoter and the CMV5 promoter by luciferase atsey.

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, embodiments of the present invention will be described in detail.

 If expression of the introduced drug resistance gene (eg, NPT gene) does not occur or is too low, the host cell into which the vector DNA has been introduced will be resistant to the corresponding drug (eg, neomycin (hereinafter NEO)). Can not get. However, the work we have done so far has shown that a large number of NEO-resistant strains were obtained in all cases using the vector DNA containing the normal NPT gene, including commercially available vectors. Was. Based on these results, the expression of the NPT gene donated from the vector DNA may serve as a selectable marker for determining whether the cells obtained as a result of transfection are transformants. However, no further effects were presumed.

 By making the induction of NPT expression extremely restrictive, the present inventors obtained NEO resistance unless the NPT gene cistern was integrated at a high level of gene expression on the host cell chromosome. It was designed so that the required level of NPT expression did not occur. This results in transformed host cells unless the integrated vector DNA is introduced into the chromosome at an extremely high level of gene expression.

NEO resistance was not obtained, and it was made impossible to survive in a medium containing a certain amount of NEO. There have been several reports using weak promoters for the NPT gene cistron of vector DNA (Mol. Cell Biol., 6, 2593, 1986; Mol. Cell Biol., 7, 1296, 1987). DNA, 7, 651, 1988). In these cases, it is unlikely that highly productive transformants have been effectively and sufficiently selected.

The principle of the present invention is that the NPT gene cyst derived from Escherichia coli transposon Tn5 In the absence of a promoter that has transcriptional activity in mammalian cells, a downstream DNA fragment containing a DNA-containing DNA fragment is downstream of a promoter that has transcriptional activity in mammalian cells followed by an optimal polyadenylation signal that has strong transcription termination activity. It significantly impairs and attenuates the expression mechanism of NPT in transformed host cells. Since the NPT gene cistron of the present invention has no promoter, it is defined as “promoterless NPT gene cistron”.

 That is, the present invention relates to a drug comprising a strong expression inducible promoter, a multiple cloning site for gene integration, and a polyadenylation signal sequence in order from the upstream, and having no downstream promoter operable in animal cells. An expression vector containing a resistance gene is provided. The expression vector of the present invention can induce high productivity of a recombinant protein in animal host cells.

 In the present invention, as the drug resistance gene, for example, a neomycin resistance gene, a hygromycin resistance gene, a zeocin resistance gene, or a blasticidin resistance gene can be used. Particularly preferred is a neomycin phosphogene. A transerase gene, most preferably a neomycin phosphotransferase gene from E. coli transposon Tn5. In addition, in the present invention, the base set forth in SEQ ID NO: 50 constructed by substituting some or all of the codons in the neomycin phosphotransferase gene with less frequently used codons in C C cells. It is also possible to construct a foreign gene expression vector by using a weakly expressed neomycin phosphotransferase gene having a sequence (also called a codon non-optimized neomycin resistance gene) as a drug resistance marker. it can.

 In the present invention, the messenger R of a very small amount of a drug resistance gene is determined by the read-through effect caused by the presence of the drug resistance gene downstream of a strong expression-inducible promoter which is blocked by a boria signal signal sequence. NA or protein is produced.

The integration of the expression vector of the present invention into a strongly expressed position on the host cell chromosome Selection of surviving strains in a medium containing the corresponding drug can be achieved due to the properties of the resistance gene cysts, but the expression of the target protein at the relevant position on the chromosome itself must be strongly induced. There is. For this reason, the promoter of the multi-cloning site (hereinafter referred to as MCS) which incorporates the protein gene and the polyadenylation signal (hereinafter referred to as polyA) are selected from those having the strongest expression inducibility. . Examples of the promoter include a human cytomegalovirus Immediate Early (hCMV MIE: Cell, 41, p. 521, 1985) promoter and β-actin promoter (Proc. Natl. Acad. Sci. USA, 84, 4831, 1987). ) Or SV40 early promoter. In the present invention, the literature of Massie B et al. (Journal of Virology, 1998 Mar; 72 (3): 2289-96. Inducible overexpression of a toxic protein by an adenovirus vector with a tetracyc 丄 ine-regulatable expression cassette. Couture F, Lamoureux L, Mosser DD, Guilbault C, Jolicoeur P, Belanger F, Langelier Y.), and using the CMV5 promoter having the nucleotide sequence of SEQ ID NO: 49 as a substitute for the CMV promoter. Thus, a foreign gene expression vector can be constructed.

 The polyA signal includes a polyA sequence derived from human growth hormone and the like. In this specification, a cistron having an MCS incorporating the protein gene of interest is referred to as an “expression cassette”.

 The present invention also relates to a transformant obtained by introducing the expression vector of the present invention into which a target gene has been incorporated into a host, and a method for producing the transformant.

The expression vector of the present invention can also preferably have a DHFR gene cistron. This expression vector uses a dihydrofolate reductase-deficient CH0 cell as a host cell, and performs efficient amplification of the gene contained in the vector DNA by methotrexate after introducing the gene of the vector DNA into the host cell. enable. In the present invention, a weakly expressed dihydrogen having the nucleotide sequence of SEQ ID NO: 51 constructed by substituting a part or all of the codons in the dihydrofolate reductase gene with low-frequency codons in CHO cells is used. leaf An exogenous gene expression vector can also be constructed by using the acid reductase gene (also called the codon non-optimized dihydrofolate reducing enzyme gene) as a selectable marker. The weakly expressed dihydrofolate reductase gene having the nucleotide sequence of SEQ ID NO: 51 is used in combination with the weakly expressed neomycin phosphotransferase gene having the nucleotide sequence of SEQ ID NO: 50. You can also.

 As for the DHFR gene cistron, a multicloning site for gene integration with a strong expression-inducible promoter without a promoter that can be expressed in mammalian cells, similar to that used for the NPT gene cistron, followed by A promoter located downstream of the optimal polyadenylation signal sequence or a promoter that has reduced expression inducibility to the DHFR gene cistron, such as the SV40 viral antigen promoter, which is normally non-infectious to CH0 cells Those from which the enhancer region has been removed are connected and used. By weakening or suppressing the inducibility of the expression of the DHFR gene in order to promote gene amplification, the present inventors can amplify the vector DNA in which MTX contained in the culture medium has been integrated into the host cell chromosome. I thought it would be more effective to guide them to encourage them.

 The present invention also includes transforming a host cell by gene transfer using a vector DNA having the DHFR gene cistron into which the protein gene has been incorporated, and amplifying the vector DNA integrated into the host cell chromosome. Provided is a method for obtaining a cell clone that produces a high level of a protein.

 The present invention further comprises transforming a host cell by introducing a gene using the above-described vector DNA into which a protein gene has been incorporated, and adapting the resulting transformant to a serum-free medium. Provided is a method for obtaining a high-level cell clone capable of stably producing a high-level protein in a medium.

The present invention also provides a vector DNA having a multicloning site for gene integration between the DHFR gene cistron and the strong expression inducible promoter and the optimal polyadduction signal. This vector DNA is ^ "It can be used for the construction of a vector DNA having a Thales NPT gene cysteine.

 In the present invention, the term "cistron" means a unit that expresses a protein by transcription / translation based on a promoter, a structural gene, and a polyadenylation signal (polyA). Any DNA sequence may be included as an import sequence. The term "strong expression inducible promoter" means a promoter generally used for protein expression in ordinary solid DNA, and is preferably a human cytomegalovirus with particularly high inducibility. -Means promoters such as Early antigen promoter,] 3-actin promoter and SV40 early promoter.

 The vector DNA of the present invention can be obtained by incorporating an NPT gene cistron and an expression cassette into a backbone vector, and if necessary, incorporating a DHFR gene cistron. There is no particular limitation on the packbone vector, and for example, a vector DNA having an E. coli replication origin (ColElori) (pUC system: Gene, 33: 103, 1985, etc.) can be used.

 As a method of using the vector of the present invention, after integrating a protein gene into the MCS of the vector of the present invention, the gene is introduced into host cells by a cationic ribosome method, and a viable strain is selected in a medium containing NEO. And a method for obtaining a cell clone that produces a high level of protein. Many of the cells obtained by selecting surviving strains in a medium containing NEO have already achieved relatively high expression levels, but select cells with higher productivity from among them For this purpose, the expression level of the protein may be measured. The resulting highly productive cells are stabilized by repeating the limiting dilution method and then subcultured. If a higher productivity strain is to be obtained, a vector having the DHFR gene cistron is used as the vector of the present invention, and MTX is added to the medium to stimulate the DHFR gene integrated into the host cell. Perform gene amplification.

Furthermore, as a more specific method of using the expression vector (pNOS-GKT2B or pNOS-GKT2B-R) of the present invention described in the Examples, a method using the following procedure is preferred. Law.

 (1) Closing the gene of the target substance (protein), and (2) incorporating it into the multicloning site (MCS) of the expression vector (pNOS-GKT2B or pNOS-GKT2B-R) of the present invention. Make an expression construct. (3) This expression construct is amplified in Escherichia coli, (4) The gene is introduced into CH0 cells (DHFR negative) by the ribofectin method or electroporation, and (5) G418 is added to the culture medium and cultured. Select resistant cells. (6) Repeat cloning while culturing the obtained cells. (7) Select clones with high production of the target substance. (8) Continue culturing until the growth rate is further increased and stabilized. (9) Gradually reduce the amount of FCS from the FCS-supplemented medium to the serum-free medium in the medium of the stabilized clone. (10) The DNA of the introduced construct was amplified by gradually adding ΜΤΧ (15ηΜ-1 μΜ) to the medium with respect to the clones with high productivity, and (11) (12) Continue culturing until the growth rate further increases and stabilizes. (13) Gradually lower the amount of FCS in the stabilized clone medium from the FCS-supplemented medium and adapt to a serum-free medium. The serum-free medium may be used in any of the steps (9) and (13).

 The following examples further specifically explain the method for producing and using the expression vector of the present invention. It is easy for those skilled in the art to replace the components such as the starting plasmid and promoter used in the present example with equivalent ones, and the scope of the present invention is not limited by the examples. Example

Example 1: Construction of the expression vector of the present invention

 (1) Preparation of neomycin resistant vector [pSV / GKT-Neo]

The NEO vector [pSV / GKT-Neo] has an E. coli replication origin (ColEl ori) derived from the plasmid pUC18 (Gene, 33, 103, 1985) and a neomycin tosphotransferase (NPT) gene cistron. A primer having the nucleotide sequence shown in SEQ ID NOs: 1 and 2 was obtained from the plasmid pUC18 by PCR using And amplified. PCR was performed under the following conditions. The PCR kit used was commercially available from Takara Shuzo, and a 10-fold concentrated reaction buffer and dNTP solution were all supplied. First, add 10-fold concentrated reaction buffer, dNTP solution, 1 μl of the primers described in SEQ ID NOS: 1 and 2 of ΙΟΟμΜ, 1 μl Ε> ΝΑ1 μl, and 0.5 μl of Pyrobest DNA polymerase (5 μ / μ1) to 36.5 μl of sterile distilled water. After the mixture was completely mixed, a PCR reaction was performed using Takara Thermal Cycler Personal. That is, three steps of denaturation (94 ° C, 30 seconds), primer annealing (55 ° C, 30 seconds), and amplification (72 ° C, 1 minute) 35 times after 94 ° (5 minutes heat treatment) The reaction was repeated at a temperature of 72 ° C. for 5 minutes to complete the reaction, and the PCR reaction described below was carried out in the same reaction cycle, comprising about 600 base pairs obtained after completion of the reaction. The PCR product was cut with (20000U / μ1), 11 and CZaI (5000U / μΐ) and Ιμΐ of restriction enzymes (all restriction enzymes described below were manufactured by NEB) at 37 ° C for 2.5 hours. After restriction enzyme treatment, the entire amount of the reaction mixture was added to a 1% agarose gel, and subjected to electrophoresis at 60 mA for 25 minutes.A DNA band consisting of about 600 base pairs was collected together with the gel and frozen at 135 ° C for 10 minutes. The DNA fragment was recovered in the supernatant after centrifugation (ColElori cassette; 36 ng / μΐ). It was carried out in the like.

Next, 3 / zg of plasmid pSV (D) S / Neo (JP-A-10-179169) was digested with 切断 μΐ of restriction enzyme & c II (20000U / μ1) and C / a I (5000υ / μ1), respectively. A DNA fragment (NE0 cassette l; 28 ng // il) consisting of about 2200 base pairs having a ΝΡΤ gene cistron downstream of the simian virus 40 (SV40) early promoter with a part removed was prepared. These two fragments were ligated with Takara DNA Ligation Kit ver.2 (Takara Shuzo) to create pSVNE0 / 0ri. Actually, 1 μΐ of ColEl ori force set and 1 μΐ of NE0 cassette 1 are mixed, and an equal amount of the mixture and Takara DNA Ligation Kit ver. 2 solution I are added and reacted at 16 ° C for 30 minutes. DNA was ligated. Next, E. coli XL-lBlue ™ combi-tent cells were transformed according to the rubidium monochloride method. Escherichia coli colonies obtained on LB-agar (Difco) containing kanamycin sulfate (SO gZml; Sigma) were converted to kanamycin sulfate. (50 μg / l; Sigma) Terrific broth (1 g of bacto-tryptone (Difco) 12 g, bacto yeast extract (Difco) 24 g, glycerol (Wako Pure Chemical Industries) 4 ml, KH2P04 2.31 g (manufactured by Wako Pure Chemical) and 12.5 g of K2HP04 (manufactured by Wako Pure Chemical). After culturing for about 20 hours, the cells were collected, and the brasmid was isolated according to the alkaline SDS method (Current Protocols in Molecular biology, pp. 1-6-3, 1986) to prepare pSVNE0 / Ori '. Ligation, transformation and plasmid isolation described hereinafter were all performed in the same manner.

Next, _P SVNE0 / 0ri 'was digested with 1 μl of restriction enzyme ^ ra I (1000 U / μ1) and Cla I (5000 U //), respectively, and the DN Α fragment consisting of about 2800 base pairs obtained by purification was obtained. (38 ng / cm2) Ligation of a linker (pSV EO linker) having multiple restriction enzyme cleavage sites obtained by annealing the newly synthesized oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 3 and 4 (pSV EO linker) After transformation and plasmid isolation, pSVNE0 / 0ri was prepared. To prepare the linker, place 50 μl each of the oligonucleotides (100 μΜ) described in SEQ ID NOS: 3 and 4 into a microtube (1.5 ml; manufactured by Tref), heat in a heat block at 70 ° C for 5 minutes, and then for about 1 hour Over 25. This was performed by cooling to C. The subsequent fabrication of the linker was performed in the same manner.

On the other hand, a DNA fragment containing a promoter sequence responsible for the induction of NPT gene cistron expression in Escherichia coli was amplified by PCR from a plasmid pSV2Neo (Stratagene) using primers having the nucleotide sequences shown in SEQ ID NOS: 5 and 6. The PCR kit used was commercially available from Takara Shuzo, and 10-fold concentrated reaction buffer, dNTP solution, and 10-fold concentrated magnesium chloride solution were all supplied as accessories. First, sterile distilled water 31.5 / 1 in 5x 10x concentrated reaction buffer, 5 μl dNTP solution, 5 / zl 10x concentrated magnesium chloride solution, 100 μl After adding 1 μl of each immobilizer, 1 μl of cyclin type 1 μl, and 0.5 / 1 of LA Tack DNA polymerase (5U / 1), and mixing them thoroughly, PCR reaction was carried out with Takaraza-Marucycler Personal. The following PCR reaction using LA Tack DNA polymerase was performed at the same mixing ratio. Approximately 400 base pairs of the resulting PCR product After developing, separating, and purifying on a gel (GKT fragment; 58 ng /// l), the product is ligated to the PCR product plasmid, pT7Blue (R) (50 ng / μ1, Novagen) by ligation. After transformation and plasmid isolation, pT7B-GKT was prepared. Then 6 mu g of _P SVNE0 / _0ri a 1 mu 1 restriction enzyme I (20000υ / μ 1) with cut-obtained E. coli replication origin, S V40 early promoter one coater removing a portion Enhansa, New [rho T A fragment consisting of approximately 2800 base pairs with a partial sequence of the gene cistron

(38 ng / ΐ) and 6 mu _§ of PT7B- GKT was obtained was cut with / ¾ί I Ν Ρ Τ gene cysts cutin fragment consisting of about 400 base pairs having a partial sequence of (GKT cassette; 9 ng / μ1) was ligated by ligation, transformed, and plasmid isolated to obtain pSV / GKT-Neo. Fig. 1 shows a flowchart of the production of [pSV / GKT-Neo].

 (2) Construction of neomycin resistant Z methotrexate resistant vector [pD]

 5 / zg of pSV / GKT-Neo described above was digested with 1 μl of restriction enzyme coR I (2000011 / μ1) Cla (5000U / μ1), and developed, separated and purified on agarose gel, about 3000 bases. A paired fragment pSV / GKT-Neo cassette (44 ng / μ1) was obtained. In addition, a part of the enhancer obtained by digestion with 1 μl of restriction enzyme ^ oR I (20000 U / μΐ) and Cla I (5000 U / 1) from pSVP (D) S / DHFR (JP-A-10-179169), respectively. A fragment consisting of about 1800 base pairs (DHFR cassette; 25 ng / μ1) having the SV40 early promoter and the dihydrofolate reductase (DHFR) gene cistron was obtained. The pSV / GKT-Neo cassette and the DHFR cassette were ligated by ligation, transformed, and plasmid isolated to obtain pND. FIG. 2 shows a flowchart of the [pND] fabrication.

 (3) Preparation of cassette plasmid [pCB] for desired gene expression

This vector consists of a promoter, a multicloning site (MCS) for integration of the protein gene of interest, and a polyadenylation signal. The promoter is derived from the human cytomegalovirus (hCMV) MIE antigen promoter, which is regarded as one of the most inducible promoters (PCMV). In addition, a pesticidal growth hormone polyA signal (bGH-polyA), which has been reported to have high productivity and excellent reproducibility, was also used for the polyA signal. First, the plasmid pRc / CMV (manufactured by Invitrogen) was amplified with PCR using LA Tack DNA polymerase with primers having the nucleotide sequences shown in SEQ ID NOs: 7 and 8, and developed, separated and purified on an agarose gel. the PCMV fragment consisting of 650 base pairs was ligated with Raigeshiyon the plasmid _P T7Blue (R), transformed, via the plasmid isolation, was created PT7B- PCMV. On the other hand, a bGH-polyA fragment consisting of about 650 base pairs amplified by PCR using LA tack DNA polymerase with primers having the nucleotide sequences shown in SEQ ID NOs: 9 and 10 from pRc / CMV was developed on an agarose gel. After isolation, purification and ligation, the plasmid was ligated to plasmid pT7Blue (R) by ligation, followed by transformation and plasmid isolation to prepare pT7B-bGH-polyA. On the other hand, 5 mu restriction plasmid pUC18 Each 1 mu 1 of _§ enzyme £ COR I and (20000U // 1) / 2dIII ( 20000ϋ /, α1) cleaved with, shown in SEQ ID NO: 1 1 and 1 2 newly synthesized A linker (CB linker) having multiple restriction enzyme cleavage sites obtained by annealing an oligonucleotide having a base sequence was ligated to prepare pUC18-CB-linker.

Next, 4.5 μg of pUC18-CB-linker consists of approximately 2700 base pairs, each of which was cleaved with 1 μl of restriction enzyme i I (20000U / μΐ) and i / jdll (20000U /// 1). The DNA fragment was developed, separated and purified on an agarose gel to obtain a PUC18-CB-linker cassette (26 ng / μΐ). Also, cutting 4 · 7 microns _§ of PT7B- PCMV with each 1 μ ΐ restriction enzyme Ab l (10000U / 1) and Ηίηά III (20000υ / 1), developed by Agarosugeru, separation, about 650 containing purified PCMV A DNA fragment consisting of base pairs (PCMV cassette; 12 ng / μ1) was obtained. Further, 1.3 μg of pT7B-bGH-polyA was digested with 1β1 restriction enzyme ο I (10000U / μΐ) BajiM I (20000υ / ^ 1), developed on an agarose gel, separated, purified and purified from about 650 bp. A DNA fragment containing bGH-polyA (bGH-polyA cassette; 2 ng / μΐ) was obtained. The PUC18-CB-linker cassette, PCMV cassette, and bGH-polyA cassette were ligated by ligation, followed by transformation and plasmid isolation to complete [pCB]. FIG. 3 shows a flowchart for preparing a cassette plasmid [pCB] for expressing a desired gene. (4) Construction of an exogenous gene expression vector [pN0S-GKT2] having an NPT gene cistron that does not have a transcriptional promoter that is active in mammalian cells

5 g of pCB is cleaved with Ιμΐ of restriction enzyme 11 I (20000υ / ^ 1) and Ηίηά III (20000ϋ / μ1), developed on agarose gel, separated and purified, and a DNA fragment pCB cassette (about 2700 base pairs) 31 η _§ / μ 1) was obtained. Then, PCR products containing the gene cistron amplified by LA-tack DNA polymerase using the primers having the nucleotide sequences shown in SEQ ID NOS: 13 and 14 were each Ιμΐ of restriction enzyme Tfc I (10000U / μ1) ≥ After digestion with NheI (5000 U / μ1), the DNA was developed, separated and purified on an agarose gel to obtain a DNA fragment NE0 cassette 2 (39 ng / μΐ) consisting of about 2700 base pairs. The pCB cassette and NE0 cassette 2 were ligated by ligation, followed by transformation, isolation of plasmid, and pCB / GKT-Neo was obtained. Furthermore, 3 _μg of pCB / GKT-Neo was digested with ΙμΙ restriction enzyme i3d III (20000υ / μ1) and ¾oR I (20000U / μ1), respectively, developed on an agarose gel, separated, purified, and PCMV, A DNA fragment (CNB cassette; 11 ng // zl) consisting of about 1000 base pairs containing the NPT gene and bGH-polyA was obtained. In addition, 5 μg of pND was cut with 1 μl of restriction enzyme S3C II (20000U / μ1), EcoR I (2000011/1) and 1 restriction enzyme sel (lOOOOOU / ^ l), respectively, and agarose gel was cut. DNA fragment consisting of approximately 2300 base pairs (DHFR cassette 2; 33 ng / μl) containing the DHFR gene cysteine containing the SV40 early promoter with the ColEl-Ori and part of the enhancer removed after development, isolation, purification. ).

 Furthermore, the newly synthesized oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 15 and 16 were annealed to obtain a linker (HSV marker) having a restriction enzyme cleavage site. 1 μl of each of the CNB cassette, DHFR cassette 2 and HS linker were ligated by ligation, and transformation and plasmid isolation were performed to prepare [pN0S-GKT2]. FIG. 4 shows a flow chart for preparing a foreign gene expression vector [pN0S-GKT2] having an NPT gene cysteine having no transcription promoter active in mammalian cells.

(5) To terminate transcription upstream of NPT gene cistron without promoter Of exogenous gene expression vector pNOS-GKT2B with bGH-polyA

PN0W3 (JP-A-10-179169) was amplified by PCR using LA Tack DNA polymerase with primers having the nucleotide sequences shown in SEQ ID NOs: 17 and 18, and developed, separated and purified on an agarose gel. Thus, a DNA fragment (bGH-polyA; 42 ng / μ1) consisting of about 200 base pairs was obtained. This was ligated to pT7Blue (R) by ligation, transformed, and plasmid isolated to prepare pT7Blue / BGHpA. 2.Cut 2 g of pT7Blue / BGHpA with 1 μl of each restriction enzyme Λ¾ίI (10000U / μ1) and I (5000U / μΐ), develop on agarose gel, separate, purify, and contain bGH-polyA DNA fragment bGH consisting of about 200 base pairs - polyA cassette 2 (3 η _§ / 1) was obtained. In addition, 5 μg of pNOS-GKT2 was cleaved with 1β1 restriction enzymes Tfci I (100000 / μ1) and Xba I (20000U / μΐ), and developed, separated and purified on an agarose gel to obtain about 4200 g of pNOS-GKT2. A DNA fragment pN0S-GKT2 cassette (13 ng / μΐ) consisting of base pairs was obtained. ΙμΙ each of the bGH-polyA cassette 2 and the pNOS-GKT2 cassette were ligated to each other, and after transformation and plasmid isolation, pNOS-GKT2B was prepared. Fig. 5 shows a flow chart of the construction of pNOS-GKT2B, an exogenous gene expression vector in which bGH-polyA for transcription termination is arranged upstream of the NPT gene cysteine without a promoter.

 (6) Construction of an exogenous gene expression vector [PN0S-GKT2B-R] with a [Egret] 3globin gene intron for improving translation efficiency

 と A heron / 3 globin gene spliced donor ^ amplified from heron genomic DNA (manufactured by CH laboratories) by PCR using LA tack DNA polymerase with primers having the nucleotide sequences shown in SEQ ID NOS: 19 and 20 The PCR product (fragment length: about 600 base pairs) of the intron-encoding region (gloSA) containing the splice receptor sequence is developed, separated and purified on an agarose gel, ligated with T7Blue (R) by ligation, and transformed. After plasmid isolation, pT7B-gloSA was prepared.

5 μg of pT7B-gloSA is digested with 1 μl of restriction enzyme I (10000 U / μ1) and Xho I (20000υ / μ1) each, developed, separated and purified on agarose gel, and contains gloSA A DNA fragment (gloSA cassette; 22 ng // zl) consisting of about 600 base pairs was obtained. In addition, 5 μg of pNOS-GKT2B was cleaved with restriction enzymes o I (100000 / μΙ) and Sal I (20000 μ / μ 1), developed, separated and purified on an agarose gel to obtain a DN consisting of about 4400 base pairs. An A fragment (pNOS-GKT2B cassette; 13 ng / μ1) was obtained. 1 μl of the gloSA cassette and the pNOS-GKT2B cassette were each ligated by ligation, transformed, and isolated to obtain pNOS-GKT2B-R.

 Fig. 6 shows a flow chart of the preparation of a foreign gene expression vector [pN0S-GKT2B-R] in which a peregrine globin gene intron for improving translation efficiency was arranged. The structure of [PN0S-GKT2B-R] is shown in FIG. 7, and the entire nucleotide sequence is shown in SEQ ID NO: 21. Example 2: Production test of human granulocyte colony-stimulating factor using the expression vector of the present invention

 HG-CSF, a kind of colony stimulating factor, was selected as the target substance for transfection of the human granulocyte coagulation stimulating factor (hG-CSF) gene with pNOS-GKT2B-R and establishment of G418-resistant early clones did. This protein has a sugar chain, and a normal recombinant protein cannot be obtained by production using Escherichia coli or yeast as a host.

 (1) Construction of hG-CSF expression vector [pN0S-GKT2B-R / hG-CSF]

Amplified from human spleen cDNA by PCR using primers having the nucleotide sequences shown in SEQ ID NOs: 22 and 23 using LA-Tack DNA polymerase, developed on an agarose gel, separated and purified, A fragment (hG-CSF fragment; 13 ng / μΐ) containing hG-CSF cDNA consisting of base pairs was obtained. This was ligated to pT7Blue (R) by ligation, transformed, and isolated with plasmid to prepare pT7Blue / hG-CSF. pT7Blue / hG-CSF is digested with 1 μl each of the restriction enzymes Tfoi I (10000U //. t1) and XbaI (20000U / ixl), developed on an agarose gel, separated, purified, and from about 600 base pairs. Thus, a DNA fragment (hG-CSF cassette; 6 ng // 1) containing cDNA of hG-CSF was obtained. In addition, pNOS-GKT2B-R is cleaved with the respective restriction enzymes ol (10000U // zl) and ¾aI (20000U /), developed, separated and purified on agarose gel, and from about 5,000 base pairs. DNA fragment (pN0S_GKT2B-R cassette; 19 η _§ / μ 1) was obtained. ΙμΙ of the hG-CSF cassette and the pNOS-GKT2B-R cassette were ligated by ligation, transformed, and isolated through plasmid to prepare pNOS-GKT2B. FIG. 8 shows a flowchart for preparing the hG-CSF expression vector [pNOS-GKT2B-R / hG-CSF], and FIG. 24 shows the entire nucleotide sequence.

 (2) Introduction of vector for expression of hG-CSF [pN0S-GKT2B-R / hG-CSF] into mammalian cells and selection of transduced strain by G418

Of 1 μ g pNOS - GKT2B- R / hG - CSF The Ripofuekuchin method 1.5 million DHFR-deficient CH0 cells (DG44 strain in 25 cm ² culture flask using (using Qiagen Efuekute down transformer Hue transfection reagent) Transferred from Dr. Gail Urlaub; Somat. Cell Mol. Genet. 12, p. 555, 1986). The method of introduction was in accordance with the manufacturer's instructions. After 48 hours, the cells are detached by trypsin treatment, and after counting the number of cells, 300,000 cells are treated with 100 ml of 0.8 mg / ml G418 in 10% dialysed fetal serum and supplemented with Iscove's Modified Dalbecco ' s After dilution in Medium, seeded in 10 96-well microtiter plates (960-well) and cultured at 37 ° C for about 3 weeks in the presence of 5% carbon dioxide, cells survived only in 117-well. (G418 resistant strain). From the surviving cells, arbitrarily select 61 G418-resistant clones, and add 1 ml of 0.8 mg / ml G418 to 10% dialysed fetal serum and add Iscove's Modified Dalbecco's Medium medium for 24 hours. The cells were transferred to an L-plate and cultured until confluent. After discarding the culture supernatant, lml of PBS (manufactured by Invitrogen) was added to each well and discarded again. To this was added 0.5 ml of a serum-free medium for CHO cells, CH0-S-SFM II (manufactured by Invitrogen), and the mixture was cultured at 37 ° C. for 72 hours in the presence of 5% carbon dioxide. Subsequently, the production amount of hG-CSF in the culture supernatant was examined.

_{(3) P N0S-GKT2B-} R / hG-CSF transduced cells clones by Hg-CSF 'production of test production of assays were carried out at a dot blot. A serum-free CHO cell culture medium for CHO cells containing 1 μl of 72-hour culture supernatant and a commercially available recombinant hG-CSF (ぺ Protech) on a nylon membrane (0.45 μm night run; Schleicher & Schuell) S- SFM II 2 × dilution series (8-0.125 ηg / μ1) and CHO-S-SFMII were separately added, and after air-drying for 1 hour and blocking with Block Ace, ίμg / μ1 A concentration of an anti-hG-CSF antibody (manufactured by Protec) followed by a horseradish peroxidase (HRP) -labeled anti-penis IgG antibody (manufactured by DAK0) was reacted. The reacted antibody was detected using a luminocapture (Ato One) by a method for detecting the activity of HRP with Super Signal West Pico Reagent (Pierce). Figure 9 shows the results obtained by the dot plot method.

 (4) Production volume of hG-CSF by pNOS-GKT2B-R / hG-CSF transduced cell clone The distribution of the expression level of each clone of hG-CSF expressed by the expression vector pN0S-GKT2B of the present invention was determined. It is shown in FIG.

 Approximately two-thirds (39 strains) of 61 G418-resistant strains at hG-CSF produced hG-CSF at significantly higher levels (0.25 μg / μ1 or more). Of the 61 strains, 33 (54.1%) had 1 μ1 or more. Even more surprisingly, 12 strains (19.7%) had ≥8 μg / μ1. The highest production level was 178 / g / ml / 3 days. This was the highest level compared with the data of the initial clone before gene amplification by a typical expression vector reported in the literature (DNA, 7, 651; 1988; Biotechnology, 10 1455, 1992; Biotechnology, 8, 662, 1990; Gene, 76, 19, 1989; Biotechnology, 9, 64, 1991).

Screening of recombinant cells by gene amplification usually takes 6 months to 1 year, and the difference in culture conditions and amplification stimulant concentration is large.The primary performance comparison of expression vectors is based on the expression of the initial clone before amplification. It is considered appropriate to perform at the level. This proved that the performance of the expression vector of the present invention was extremely high. As a result, the expression vector with the promoter-less NPT gene cistron blocked by a strong polyadenylation signal is extremely low in the number of G418-resistant strains obtained, while at the same time, it can produce the target protein with high efficiency. It was confirmed that a sex cell line could be established. This proved that the expression vector of the present invention enables very high protein expression levels. Example 3: Gene amplification of G418 resistant cell line

 Among the G418-resistant cell lines, clones with hG-CSF production of 77 μg / ml / 3 days were subcultured and stabilized, and then gene amplification by MTX was performed. After initial amplification for 2 weeks in a medium containing 15 nM MTX, 2 weeks in a medium containing 60 nM MTX, 2 weeks in a medium containing 250 nM MTX, and a further 2 weeks in a medium containing 1 μM MTX, hG -The production level of CSF increased to 117 // g / ml / 3 days per medium. The concentration of MTX used for gene amplification is generally multi-step between ΙΟηΜ and ΙΟμΜ, and 最終 μΜ is often used as the final concentration.However, due to the toxicity of the cells, exposure to high concentrations may result in stable recombinant cell lines. There is a problem with establishment. For this reason, achieving high productivity at low concentrations was also an important evaluation criterion, and was set to 1 / ζ in this experiment. In addition, the exposure period, including normal selection, was 6 to 12 months, whereas this experiment was performed in about 8 weeks. Despite these experimental conditions, an effective increase in hG-CSF production was observed, confirming the high performance of the expression vector in gene amplification systems.

Example 4: Expression of recombinant human G-CSF using pNOS

 (1) Preparation of pN0S / hG-CSF expression vector

 An expression vector pNOS was constructed in the same manner as in Example 1, except that Tn5 derived from Escherichia coli was directly used as the NE0 resistance gene, and HGHpA was used instead of BGHpA. Further, according to the method described in Example 2 (1), an expression vector pNOS / hG-CSF was constructed by inserting a human granulocyte colony-stimulating factor (hG-CSF) gene into the cloning site. The structure of pN0S / hG-CSF is shown in FIG. 11, and the entire nucleotide sequence is shown in SEQ ID NO: 25.

 (2) Measurement of the expression level of recombinant human G-CSF using pNOS

25cm when ² flask culture to have CH0 cells 4 0~ 8 0% Confluent state, using a transformer Hue transfection reagent sold by Qiagen company, incorporates a pNOS-humanG-CSF expression vector to CH0 cells It is. After 2 days, trypsinize, detach cells from culture flask, check cell count using hemocytometer, The cells were spread on 10 96-well plates at a density of ⁵ × 10 ⁵ cells / 100 ml. Two weeks later, the number of emerging colonies was counted, and 146 colonies were confirmed. Transfer 72 single colonies appearing in the 10 x 96 well plate to the 24 well plate. When the clone transferred to the 24-well plate became confluent, it was washed once with 1 XPBS, and then a serum-free medium was added, and the culture supernatant was collected 3 days later.

 After 3 days, the culture supernatant and the dilution series standard were spotted on the membrane one by one. After air drying for 1 hour, blocking was performed. Then, it was washed once with TBS / T. After the washing was completed, a primary antibody reaction was performed with 1 jug / ml of rabbit-anti human G-CSF antibody. Again, it was washed three times with TBS / T. A secondary antibody reaction with HRP-anti-rabbit IgG was performed. After washing again with TBS / T three times, the expression level of human G-CSF was confirmed by luminescence reaction with Super Signal West Pico reagent. FIGS. 12 and 13 show examples of detection of hG-CSF expressed by the expression vector pNOS / human G-CSF of the present invention by dot blotting. Fig. 14 shows the distribution of the expression level of each clone of hG-CSF expressed by pNOS / human G-CSF.

 As a result of confirming the expression level of human G-CSF using the pNOS expression vector by dot blot (46 clones tested for concentration), pNOS contained more than 65% of the clones exceeding 1 g / ml. Of which 33% showed an expression level exceeding 8 g / ml. Example 5: Construction of CMV5 promoter (Figures 15 and 16)

(1-1) PCR of L1 region was performed from adenovirus type2 genome ^ l. (The PCR kit is commercially available from Takara Shuzo, and all buffer 'dNTPs used were supplied.) First, add 2.5 μM dNTP 5 μl, 10 x Pyrobest PCR buffer 5 μl to 36.5 μl of sterile distilled water, and add L1 sense primer region [SEQ ID NO: 2 6] (ΙΟΟμΜ) Ιμΐ and antisense primer [SEQ ID NO: ₂ 7] _(ΙΟΟμΜ) Ιμΐ, the DNA Imi and铸型, there DNA poly camera. 0.5 µl of Pyrobest (5υ / μ1) was added, mixed by pipetting, and PCR was performed under the following conditions. That is, after heating at 94 ° C for 5 minutes, 94 ° C for 30 seconds, 55 ° C After repeating three steps of 30 seconds and 72 ° C for 30 seconds 35 times, the reaction was terminated by treating at 72 ° C for 5 minutes. After completion of the PCR, the amplification of the target gene region was confirmed by electrophoresis using a 1% agarose gel. As a result, a band was observed around 40 bp.

(1-2) L2 region PCR was performed from adenovirus type2 .genome {μ}. (The PCR kit is commercially available from Takara Shuzo, and all buffer 'dNTPs used were supplied.) First, add 5 μl of 2.5 mM dNTPs and 5 μl of 10 x Pyrobest PCR buffer to 36.5 μl of sterilized distilled water, and add L2 Using the sense primer [SEQ ID NO: 28] (ΙΟΟμΜ) Ιμΐ and the antisense primer [SEQ ID NO: 29] (ΙΟΟμΜ) Ιμ DNA and DNA Ιμ の in the region, the DNA polymerase .Pyrobest (5 υ / μ1) was added there. .5 µl was added, pitting was performed, and the mixture was thoroughly mixed. PCR was performed under the following conditions. That is, after the heat treatment at 94 ° C for 5 minutes, three steps of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds were repeated 35 times, and the reaction was completed by treating at 72 ° C for 5 minutes. After completion of PCR, amplification of the target gene region was confirmed by electrophoresis using 1% agarose gel, and a band was confirmed around 70 bp.

(1-3) PCR of the L3 region was performed from adenovirus type2 genome {μ}. (The PCR kit is commercially available from Takara Shuzo and all buffer 'dNTPs used were supplied.) First, add 2.5 µdNTP 5 µl, 10 x Pyrobest PCR buffer 5 µl to 36.5 µl of sterile distilled water, and add L3 Sense primer [SEQ ID NO: 30] (ΙΟΟμΜ) Ιμΐ and antisense primer [SEQ ID NO: 3 1] (ΙΟΟμΜ) 1μ1 and DNA Ιμ 铸 are used as the type, and the DNA polymerase 'Pyrobest (5ϋ / μ1) .5μ1 was added, mixed by pipetting, and PCR was performed under the following conditions. That is, after the heat treatment at 94 ° C for 5 minutes, three steps of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds were repeated 35 times, and the reaction was completed by treating at 72 ° C for 5 minutes. After completion of the PCR, the amplification of the target gene region was confirmed by electrophoresis using a 1% agarose gel. As a result, a band was confirmed at around 200 bp.

(1-4) PCR was performed on Rl, 2, 3 regions from adenovirus type2 genome {μ}. (The PCR kit is sold by Takara Shuzo and all of the buffer 'dNTPs used were attached.) First, add 5 μl of 2.5 mM dNTP, 5 μl of 10 x Pyrobest PCR buffer to 36.5 μl of sterilized distilled water, and add Rl, Sense primers of two or three regions [SEQ ID NO: 32] (ΙΟΟμΜ) Sense primer [SEQ ID No. 3 3] (ΐΟΟμΜ) Ιμΐ, DNA Ιμ 铸 was made into 铸 type, DNA polymerase 'Pyrobest (5υ / μ1) was added to it, 0.5μ1 was added, and mixed by pipetting. PCR was carried out. That is, after the heat treatment at 98 ° C for 1 minute, three steps of 98 ° C for 5 seconds, 55 ° C for 20 seconds, and 72 ° C for 15 seconds were repeated 35 times, followed by treatment at 72 ° C for 2 minutes to terminate the reaction. After completion of the PCR, amplification of the target gene region was confirmed by electrophoresis using a 1% agarose gel, and a band could be confirmed near lOObp. (1-5) ρΟίνΤηΤ (10ηΕ / μ1) PCR of the CMV promoter region was performed using 1 μ1 as type I. (The PCR kit is commercially available from Takara Shuzo and buffer 'dNTP'MgCl2 was all used.) First, 2.5 mM dNTPs 5 μl, 25 mM MgCl2 5 μl, 10 χ LA PCR buffer in 31.5 μl of sterile distilled water 5μ1 was added, and the CMV promoter sense primer [SEQ ID NO: 34] (ΙΟΟμΜ) Ιμΐ and antisense primer [SEQ ID NO: 35] (ΙΟΟμΜ) 1μ1 and DNA

Was added and mixed by pipetting to perform PCR under the following conditions. That is, after the heat treatment at 94 ° C for 5 minutes, the three steps of 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds are repeated 35 times, followed by treatment at 72 ° C for 5 minutes to terminate the reaction. did. After completion of the PCR, the amplification of the target gene region was confirmed by electrophoresis using 1% agarose gel. As a result, a band was confirmed at around 650 bp. [SEQ ID NO: 4 1]

 (1-6) Add 3M NaoAc-δμΙ and 100% ethanol · 125μ1 to the PCR reaction solution of (1-1), and centrifuge at 14000rpm for 10 minutes. After the centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire volume of this solution to a 1% agarose gel, and electrophorese at 50 mA for 25 minutes. A band was confirmed at around 40 bp when compared to the DNA marker. The band was collected and frozen at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. The obtained extract was designated as L1 (extraction concentration = 82 ng / Vl).

(1-7) Add 3M NaoAc-δμΙ and 100% ethanol · 125μ1 to the PCR reaction solution of (1-2), and centrifuge at 14000rpm for 10 minutes. After the centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. This solution in 1% agarose gel Apply the whole amount and perform electrophoresis at 50mA for 25 minutes. A band was found around 70 bp when compared with the DNA marker. The band was collected and frozen for -135> 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. The obtained extract was designated as L2 (extraction concentration = 104 _ng / l).

(1-8) Add 3M NaoAc-δμΙ and 100% ethanol · 125μ1 to the PCR reaction solution of (1-3), and centrifuge at 14000rpm for 10 minutes. After the centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire volume of this solution to a 1% agarose gel, and electrophorese at 50 mA for 25 minutes. A band was confirmed at around 200 bp when compared with the DNA marker. The band was recovered and frozen at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. Extract the obtained extract into L3 (extraction concentration

And

(1-9) Add 3Μ NaoAc-δμΙ and 100% ethanol · 125μ1 to the PCR reaction solution of (1-4) and centrifuge at 14000rpm for 10 minutes. After the centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire volume of this solution to a 1% agarose gel, and electrophorese at 50 mA for 25 minutes. A band was confirmed around lOObp as compared with the DNA marker. The band was collected and frozen at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. The obtained extract was designated as R123.

(1-10) Add 3M NaoAc-δμΙ and 100% ethanol · 125μ1 to the PCR reaction solution of (I-5), and centrifuge at 14000rpm for 10 minutes. After the centrifugation, the pellet was confirmed and the supernatant was gently removed. Add LxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire volume of this solution to a 1% agarose gel, and electrophorese at 50 mA for 25 minutes. A band was found around 650 bp when compared with the DNA marker. The band was collected and frozen at -135 ° Ο for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. The obtained extract was designated as PCMV (extraction concentration = 54η _§ / μ1).

(1-11) PCR of the L123 region was performed using {μ} of each of the extracts of 1-6, 7, and 8. (The PCR kit is sold by Takara Shuzo and all of the buffer 'd TP' MgCk is attached. Product was used. First, add 5 μl of 2.5 mM dNTP, 5 μl of 25 mM MgCl2, and 5 μl of 10 x LA PCR buffer to 29.5 μl of sterile distilled water, and add a sense primer for the LI region [SEQ ID NO: 26] (ΐΟΟμΜ) and an antisense primer for the L3 region. [SEQ ID NO: 3 1] (ΙΟΟμΜ) Ιμΐ, total 3μ1 of DNA, type 錶, add 0.5μ1 of DNA polymerase * LA-Taq (5ϋ / μ1), mix by pipetting, and mix thoroughly PCR was performed. That is, after the heat treatment at 94 ° C for 5 minutes, three steps of 94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 30 seconds were repeated 35 times, and then the reaction was terminated by treating at 72 ° C for 5 minutes. After completion of PCR, amplification of the target gene region was confirmed by electrophoresis using 1% agarose gel, and a band was confirmed around 300 bp.

(1-12) 3M NaoAc-δμΙ and 100 ° / in the PCR reaction solution of 1-11. Add ethanol · 125μ1 and centrifuge at 14000rpm for 10 minutes. After the centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire volume of this solution to a 1% agarose gel and electrophorese for 50πιΑ for 25 minutes. Compared to the DNA marker, a band around 300 bp was confirmed. The band was collected and frozen at -135 ° Ο for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. L123

And [Sequence number 4 2]

(1-13) PCR of the R123 'region was performed using the extract of 1-9. (The PCR kit was sold by Takara Shuzo and all buffer 'dNTPs used were supplied.) First, add 5 μl of 2.5 mM dNTPs and 5 μl of 10 x Pyrobest PCR buffer to 36.5 μl of sterilized distilled water, and add R123 The region's sense primer [SEQ ID NO: 32] (ΙΟΟμΜ) Ιμΐ and antisense primer [SEQ ID NO: 36] (ΙΟΟμμ) 1μ1 and DNA 錄 μ 錄 were type I, and DNA polymerase 'Pyrobest (5υ / μ1) was added to it. 5 µl was added, pipetting was performed, and the mixture was completely mixed. PCR was performed under the following conditions. That is, after the heat treatment at 98 ° C. for 1 minute, three steps of 98 ° C. for 5 seconds, 55 ° C. for 20 seconds, and 72 ° C. for 15 seconds were repeated 35 times, and then the reaction was terminated at 72 ° C. for 2 minutes. After completion of the PCR, amplification of the target gene region was confirmed by electrophoresis using a 1% agarose gel, and a band was confirmed at around 150 bp. (1-14) Add 3M NaoAc-δμΙ and 100% ethanol · 125μ1 to the PCR reaction mixture of 1-13, and centrifuge at 14000rpm for 10 minutes. After centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire amount of this solution to a 1% agarose gel, and run 50 mA 'for 25 minutes. A band was confirmed around 150 bp when compared with the DNA marker. The band was collected and frozen at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. The obtained extract was R123- 1 (extract concentration = 107η δ / _μ1).

(1-15) Using the extract of 1-14, PCR was performed on the R123 "region. (The PCR kit is commercially available from Takara Shuzo and all buffer 'dNTPs used were supplied.) First, 2.5 mM dNTP 5 μl and 10 x Pyrobest PCR buffer 5 μ1 were added to 36.5 μl of sterile distilled water. The R123 region sense primer [SEQ ID NO: 32] (ΙΟΟμΜ) Ιμΐ and the antisense primer [SEQ ID NO: 37] (ΙΟΟμΜ) 1μ1, DNA Ιμ 铸, and the DNA polymerase 'Pyrobest (5ϋ / μ1) was added to 0.5μ1 and pipetting was performed to mix thoroughly, and PCR was performed under the following conditions. That is, after the heat treatment at 98 ° C for 1 minute, three steps of 98 ° C for 5 seconds, 55 ° C for 20 seconds, and 72 ° C for 15 seconds were repeated 35 times, and then the reaction was completed at 72 ° C for 2 minutes. After completion of the PCR, the amplification of the target gene region was confirmed by electrophoresis using a 1% agarose gel, and a band was confirmed at around 150 bp.

(1-16) Add 3M NaoAc-δμΙ and 100% ethanol · 125μ1 to the PCR reaction mixture of 1-15 and centrifuge at 14000rpm for 10 minutes. After the centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire volume of this solution to a 1% agarose gel, and electrophorese at 50 mA for 25 minutes. Compared to the DNA marker, a band around 150 bp was confirmed. The band was collected and frozen at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. Extract the obtained extract into R123-2 (extraction concentration

And [SEQ ID NO: 4 3]

(1-17) Mix PCR extract of 1-12 (extraction concentration ng / μΐ) Ιμΐ and pT7Blue Ιμΐ (Novagen 50η / μ1). There is an equal amount of ligation kit ver. 2 solution I (Takara Shuzo Co., Ltd. Was added and a ligation reaction was performed at 12 ° C. for 2 hours. Thereafter, a transformant of E. coli was obtained. Thereafter, a sequence was performed to select a plasmid having no base substitution, and the resulting plasmid was designated as pT7B-L123.

(1-18) PCR-derived extract of 1-16 (extracted concentration ng / μΐ) Ιμΐ and pT7Blue Ιμΐ (Novagen Inc. · 50η β _/ μ1) mixing. An equal volume of ligation kit ver. 2 solution I (Takara Shuzo Co., Ltd.) was added thereto, and a ligation reaction was performed at 12 ° C. for 2 hours. Thereafter, a transformant of E. coli was obtained. Thereafter, a sequence was performed to select a plasmid without base substitution, and the resulting plasmid was designated as pT7B-R123.

 (1-19) Mix 1-10 PCR-derived extract (extraction concentration -ng / μΐ) Ιμΐ and p Blue Ιμΐ (Novagen, 50η§ / μ1). An equal amount of ligation kit ver. 2 solution I (Takara Shuzo Co., Ltd.) was added thereto, and a ligation reaction was carried out for 12 ° 02 hours. Thereafter, a transformant of E. coli was obtained. Thereafter, a sequence was performed to select a plasmid without base substitution, and the resulting plasmid was designated as T7B-CMV.

 (1-20) PCR of the L123 'region was performed using pT7B-L123 (lOng / μΙ) of 1-17 as a 铸 type. (The PCR kit was sold by Takara Shuzo and all the buffer 'dNTPs were used as accessories.) First, add 2.5 µm dNTPs 5 µl, 10 x Pyrobest PCR buffer 5 µl to 36.5 µl of sterile distilled water, and add L123 Region sense primer [SEQ ID NO: 26] (ΙΟΟμΜ) Ιμΐ and antisense primer [SEQ ID NO: 38] (ΐΟΟμΜ) Ιμΐ, DNA Ιμΐ, and DNA polymerase 'Pyrobest (51Ι / μ1) .5μ1 was added, mixed by pipetting, and PCR was performed under the following conditions. That is, after the heat treatment at 94 ° C for 5 minutes, three steps of 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds were repeated 35 times, followed by treatment at 72 ° C for 5 minutes to terminate the reaction. After completion of PCR, amplification of the target gene region was confirmed by electrophoresis using 1% agarose gel, and a band was confirmed around 300 bp.

 (1-21) PCR of the R123-IgG3 'region was performed using pT7B-R123 (lOng / μΙ) of 1-18 as type II.

(The PCR kit is sold by Takara Shuzo and all buffers * dNTPs were used.) First, 2.5 mM dNTPs 5 μ1 10 x Pyrobest PCR buffer in 36.5 μl of sterile distilled water 5μ1 was added, and the R123 region sense primer [SEQ ID NO: 39] (ΜμΜ) Ιμΐ and antisense primer [SEQ ID NO: 40] (ΜμΙ) Ιμΐ, DNA Ιμΐ were made into 鎵 type, and the DNA polymerase 'Pyrobest (5υ / μ1) was added and mixed by pipetting, and PCR was performed under the following conditions. That is, after the heat treatment at 94 ° C for 5 minutes, three steps of 95 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 30 seconds were repeated 35 times, followed by treatment at 72 ° C for 5 minutes to terminate the reaction. After completion of the PCR, the amplification of the target gene region was confirmed by electrophoresis using a 1% agarose gel, and a band was confirmed at around 200 bp.

(1-22) Add 3M NaoAc-δμΙ and 100% ethanol · 125μ1 to the 1-20 PCR reaction mixture and centrifuge at 14000rpm for 10 minutes. After centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire amount of this solution to a 1% agarose gel, and electrophorese at 50 mA for 25 minutes. A band was confirmed around 300 bp when compared with the DNA marker. The band was collected and frozen at -135 ° C for 10 minutes. Then centrifuge at 14000rpm 'for 10XII. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. Extract the obtained extract into L123-1

And [SEQ ID NO: 4 4]

(1-23) Add 3M NaoAc-δμΙ and 100% ethanol · 125μ1 to the PCR reaction solution of 1-21 and centrifuge at 14000rpm for 10 minutes. After centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire amount of this solution to a 1% agarose gel, and run 50 mA 'for 25 minutes. A band was confirmed around 300 bp when compared with the DNA marker. The band was collected and frozen at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml Eppendorf Tude. The resulting extract R123 - was IgG3 (extract concentration = 79η δ / _μ1). [Sequence number 4 5]

(1-24) PCR of the L123-R123-IgG3 'region was performed using Ιμΐ of each of the extracts of 1-22 and 23. (PCR kit was used all in those sold buffer 'dNTP'MgCl2 is the attached article from Takara Shuzo.) First, sterile distilled water 2 ^9. 5μ1 to 2. 5mM dNTP 5μ1, 25mM Add 5 μl of MgCl2 5 μl of 10 x LA PCR buffer, and add the sense primer [SEQ ID NO: 1] (ΙΟΟμΜ) Ιμΐ in the LI region and the antisense primer of R123-IgG3 '[SEQ ID NO: 15] (ΙΟΟμΜ) Ιμΐ, DNA total 3μ1. 0.5 μl of DNA polymerase 'LA-Taq (5 U / l) was added thereto, mixed by pitting and mixed thoroughly, and PCR was performed under the following conditions. That is, after the heat treatment at 98 ° C for 1 minute, three steps of 98 ° C for 5 seconds, 55 ° C for 20 seconds, and 72 ° C for 15 seconds were repeated 35 times, and then the reaction was terminated by treating at 72 ° C for 2 minutes. . After completion of the PCR, the amplification of the target gene region was confirmed by electrophoresis using a 1% agarose gel, and a band was confirmed at around 500 bp. [SEQ ID NO: 4 6]

(1-25) Add 3M NaoAc-δμ に to the PCR reaction mixture of 1-24 and 100 ° /. Add ethanol · 125μ1 and centrifuge at 14000rpm for 10 minutes. After centrifugation, the pellet was confirmed and the supernatant was gently removed. Add lxDSB (ΙΟμΙ) to completely dissolve the pellet. Apply the entire volume of this solution to a 1% agarose gel, and electrophorese at 50 mA for 25 minutes. Compared to the DNA marker, a band was confirmed at around 500bp. Collect the band and freeze it at -135 ° C for 10 minutes. Then centrifuge at HOOOrpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. The obtained extract was LR123 (extract concentration = 56η β / _μ1).

(1-26) 1-25 PCR

Mix Ιμΐ and pT7Blue Ιμΐ (Novagen 50η _§ / μ1). An equal amount of ligation kit ver. 2 solution I (Takara Shuzo Co., Ltd.) was added thereto, and a ligation reaction was carried out for 12 ° 02 hours. Thereafter, a transformant of E. coli was obtained. Thereafter, a sequence was performed to select a plasmid having no base substitution, and the resulting plasmid was designated as ρ {7} -LR123.

(1-26) 1-25 and the obtained plasmid * 2. Use the 5 [mu] _beta, 25 ° with the restriction enzyme (Nyuipushironbeta Co.) BamH I Ιμΐ (20000U / ml ) and EcoR I Ιμΐ (20000U / ml) C- After digestion with 0 / N, LR123 was cut out. After restriction enzyme treatment, the reaction mixture (液 μΙ) was applied to a 1% agarose gel, and electrophoresed at 50 mA for 25 minutes. As a result, bands were confirmed at around 2800 bp and 400 bp when compared with the DNA marker. The entire reaction mixture was applied to 1% agarose gel again, and after electrophoresis at 50 mA for 25 minutes, a band that could be confirmed at around 400 bp was recovered. The recovered gel extract was frozen at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. Transfer the centrifuged supernatant to a new 1.5 ml Transferred to an Appendorf tube. The obtained extract was designated as LR123 digest (extraction concentration = 16η _§ / μ1).

(1-28) Plasmid obtained in 1-19 · Using 2.5μ _§ , use restriction enzyme (ΝΕΒsha) Hind III Ιμΐ (20000U / ml) and Bgl II Ιμΐ (lOOOOU / ml) at 25 ° C ' The CMV promoter was cut out by digestion with 0 / N. After restriction enzyme treatment, the reaction mixture (反 応 μΙ) was applied to 1% agarose gel, and electrophoresed at 50 mA 'for 25 minutes. As a result, bands were confirmed at around 2800 bp and 650 bp when compared with the DNA marker. The entire reaction solution was applied to 1% agarose gel again, and 50 mA * electrophoresis was performed for 25 minutes, after which a band that could be confirmed at around 650 bp was recovered. Freeze the recovered gel extract at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was renewed and transferred to a 1.5 ml Eppendorf tube. The obtained extract was used as CMV promoter digest (extraction concentration = 14 ng / l).

(1-29) pCB · 2. using 5μ _§, 25 ° 0 / N was digested with restriction enzymes (Nyuipushironbeta Co.) Hind III Ιμΐ (20000U / ml ) and Not I Ιμΐ (10000U / ml) . After restriction enzyme treatment, apply the entire reaction mixture to 1% agarose gel, and perform electrophoresis at 50 mA for 25 minutes. A band was found around 3500 bp when compared to the DNA marker, and the band was collected. Freeze the recovered gel extract at -135 ° C for 10 minutes. Then centrifuge at OOOrpm 'for about 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. The obtained extract was used as pCB digest (extraction concentration = 30 ngl).

(1-30) pCB digest obtained in 1-29 (30η _§ / μ1) Ιμΐ, LR123 digest obtained in 1-26 (ieng / μΐ) Ιμΐ, CMV promoter digest obtained in 1-27 (14ng / Vl) Ιμΐ, and add an equal volume (3μ1) of ligation kit ver. 2 solution I (Takara Shuzo Co., Ltd.), perform a ligation reaction at 16 ° 030 minutes, and then perform transformation of Escherichia coli Got a body. The plasmid obtained from the transformant was named pCMV5. [Sequence number 47] Example 6: Preparation of pCMV5-lucif erase (Fig. 17)

(II-1) Using pCMV5 · 2.5μ _§ , restriction enzyme (Isha) Not I Ιμΐ (10000U / ml) and Xba Digestion was performed at 25 ° C · 0 / I Iμΐ (20000U / ml). After restriction enzyme treatment, apply the entire reaction solution to 1% agarose gel, and perform electrophoresis at 50 mA for 25 minutes. A band was found around 4000 bp when compared to the DNA marker, so the band was collected. Freeze the recovered gel extract at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml eppendorf tube. The resulting extract pCMV5 was digest (extract concentration = 19 _n gl).

(II-2) Using pCB-luc if erase2.5 μ _§ , use restriction enzymes (NEB) Not I Ιμΐ (lOOOOOU / ml) and Xba I Ιμΐ (20000U / ml) at 25 ° C '0 / N Digested. Apply the reaction solution 1 _mu 1 1% Agarosugeru after restriction enzyme treatment, 50 mA '25 min electrophoresis result, a band was confirmed to 3500bp and 1600bp Atari when compared to DNA markers. The entire amount of the reaction solution was applied again to a 1% agarose gel, and a band that can be confirmed at around 1600 bp was collected after electrophoresis at 50 mA for 25 minutes. Freeze the recovered gel extract at -135 ° C for 10 minutes. Then centrifuge at 14000 rpm for 10 minutes. The centrifuged supernatant was transferred to a new 1.5 ml Eppendorf buffer. The resulting extract was defined as luciiferase gene digest (extraction concentration = 27η _§ / μ1). [SEQ ID NO: 4 8]

(II-3) pCMV5 digest (19η _§ / μ1) Ιμΐ obtained in II-1 and luciferase gene digest (27ng ^ l) Ιμΐ obtained in II-2 were mixed, and the same amount (2μ1) Ligation kit ver. 2 solution I (Takara Shuzo Co., Ltd.) was added, a ligation reaction was performed at 16 ° C for 30 minutes, and then E. coli was transformed to obtain a transformant. The plasmid obtained from the transformant was designated as pCMV5-luciferase. Example 7: Activity measurement of CMV5 promoter by luciferase assay

(III-1) In order to measure the activity of the CMV5 promoter using CH0DG44-S and HE293RT, each was cultured in a 25 cm ² flask. On the day of transfection, the cells cultured in a 25 cm ² flask are centrifuged in a 15 ml centrifuge tube. After centrifugation, discard the culture supernatant, and add 5 ml of CH0-S-SFM II medium (GI BC0) containing 100 mM hypoxanthin and 10 mM thymidine. Add 5 ml of Free Style medium (GIBC0) to the other. After counting both cells, 24 Cells were added to each well plate so that each well contained 2.5 105 cells I well. (II 1-2) 0.3 μl of each medium was added to 61 μl of a transfection reagent (QIAGEN) containing 166 ng of DNA (pCMV5-luciferase) per well, added to a 24-well plate, and cultured for 48 hours did.

 (III-3) After 48 hours, collect the cells in a 1.5 ml tube and discard the supernatant. Then, add PLD-30 (reagent attached to the Pitka Gene Dual Sea Pansy Kit, Toyo Ink Manufacturing Co., Ltd.) in a volume of ΙΟΟμΙ, and stir gently for 15 minutes. After 15 minutes, centrifuge at 200 xg for 10 minutes. When 5 μl of the centrifuged supernatant was added to 195 μl of PLD-30 reagent and lucif erase assay was performed with a fluorescence measurement device (ATT0: Noreluminescence Sensor PSN · R), stronger activity was confirmed than that of the CMV promoter. The results are shown in FIG. Industrial applicability

 According to the present invention, it is possible to provide an expression vector capable of producing a high-level recombinant protein using a mammalian cell as a host.

Claims

-The scope of the claims

1. Includes, in order from the upstream, a strong expression inducible promoter, a multi-cloning site for gene integration, and a polyadenylation signal sequence, and a drug resistance gene having no promoter operable in animal cells downstream thereof. An expression vector that induces high productivity of a recombinant protein in animal host cells.

 2. The expression according to claim 1, wherein the strong expression inducible promoter is a human cytomegalovirus Majorlmmeidately-Early antigen promoter, CMV5 promoter (synthetic chimera one promoter), actin promoter or SV40 early promoter. vector.

 3. The method according to claim 1, wherein the drug resistance gene is a neomycin resistance gene, a codon non-optimized neomycin gene, a hygromycin resistance gene, a zeocin resistance gene, or a blasticidin resistance gene. Expression vector.

 4. The expression vector according to any one of claims 1 to 3, wherein the drug resistance gene is a neomycin phosphotransferase gene.

 5. The expression vector according to any one of claims 1 to 4, wherein the drug resistance gene is a neomycin phosphotransferase gene derived from Escherichia coli transposon ηη5.

 6. A trace amount drug-resistance gene messenger RNA or protein is generated by the read-through effect due to the presence of the drug resistance gene downstream of the strong expression inducible promoter blocked by the polyadenylation signal sequence. Item 6. The expression vector according to any one of Items 1 to 5.

 7. The downstream of the drug resistance gene further comprises a dihydrofolate reductase gene without a promoter operable in animal cells or a codon non-optimized dihydric folate reductase gene. An expression vector according to any one of the above.

8. In the downstream of the drug resistance gene, a strong expression-inducing promoter A dihydrofolate reductase gene or codon non-optimized dihydrofolate, which contains a promoter, a multicloning site for gene integration, and a polyadenylation signal sequence and has no downstream promoter operable in animal cells The expression vector according to any one of claims 1 to 7, having an original enzyme gene.

 9. The expression according to any one of claims 1 to 6, wherein the expression has a dihydrofolate reductase gene or a codon non-optimized dihydrofolate reductase gene downstream of the promoter with low expression inducibility. vector.

 10. Promoter derived from a protein gene whose expression is low in expression is normally expressed only in a very small amount in mammalian cells, promoter derived from a virus antigen gene which is less susceptible to mammals, or enhancer sequence from the above promoter. The expression vector according to any one of claims 7 to 9, which is a promoter from which has been removed.

 11. Recombinant expression vector obtained by incorporating a target gene into the multi-jungle site for gene integration of the expression vector according to any one of claims 1 to 6.

 12. A recombinant expression vector obtained by incorporating a target gene into a multicloning site for gene integration of the expression vector according to any one of claims 7 to 10.

 13. A transformant having the expression vector according to any one of claims 1 to 10 or the recombinant expression vector according to claim 11 or 12.

 14. A method for producing a transformant that highly expresses a target gene, comprising introducing the recombinant expression vector according to claim 11 or 12 into a host animal cell.

 15. A transformant is obtained by introducing the recombinant expression vector according to claim 11 or 12 into a host animal cell, and acclimating the obtained transformant to a serum-free medium. A method for obtaining a transformant capable of stably producing a protein in a serum-free medium.

16. The recombinant expression vector according to claim 12 is deficient in dihydrofolate reductase. A method for amplifying a target gene, comprising introducing into a damaged CHO cell and then culturing the cell in the presence of methotrexate.